biostudies-literatureHwang JGouvernement du Canada | Natural Sciences and Engineering Research Council of Canada (Conseil de Recherches en Sciences Naturelles et en Génie du Canada)New Brunswick Innovation Foundation (Fondation de l'innovation du Nouveau-Brunswick)National Science Foundation (NSF)10495https://www.ebi.ac.uk/biostudies/studies/S-EPMC12647599biostudies-literatureUnknown16(1)The inversion of C3 stereochemistry in monoterpenoid indole alkaloids (MIAs), derived from the central precursor strictosidine (3S), is essential for producing pharmacologically important 3 R MIAs and spirooxindoles such as reserpine. While early MIA biosynthesis preserves the 3S configuration, the mechanism underlying C3 inversion has remained unresolved. Here, we identify and biochemically characterize a conserved oxidase-reductase pair in Gentianales: heteroyohimbine/yohimbine/corynanthe C3-oxidase (HYC3O) and C3-reductase (HYC3R), which together invert the 3S stereochemistry to 3 R across diverse substrates. HYC3O and HYC3R are encoded within biosynthetic gene clusters in Rauvolfia tetraphylla and Catharanthus roseus, homologous to a geissoschizine synthase (GS) cluster also uncovered.Nature communicationsAncient gene clusters govern the initiation of monoterpenoid indole alkaloid biosynthesis and C3 stereochemistry inversion.PMC126475992030871RAI_2023_054, RPI_2022_002RGPIN-2020-04133Fleck SJKirshner JMai ZDeslongchamps GMann SGABlight BASilliphant SNShahsavarani MAlbert VAHwang JEnglehart SAGao DRichardson MBLian JPerley JOGuo JDeschenes DARDoiron SSSeveck ADQu YCalhoun LGarza-Garcia JJOfalseAncient gene clusters govern the initiation of monoterpenoid indole alkaloid biosynthesis and C3 stereochemistry inversion.The inversion of C3 stereochemistry in monoterpenoid indole alkaloids (MIAs), derived from the central precursor strictosidine (3S), is essential for producing pharmacologically important 3 R MIAs and spirooxindoles such as reserpine. While early MIA biosynthesis preserves the 3S configuration, the mechanism underlying C3 inversion has remained unresolved. Here, we identify and biochemically characterize a conserved oxidase-reductase pair in Gentianales: heteroyohimbine/yohimbine/corynanthe C3-oxidase (HYC3O) and C3-reductase (HYC3R), which together invert the 3S stereochemistry to 3 R across diverse substrates. HYC3O and HYC3R are encoded within biosynthetic gene clusters in Rauvolfia tetraphylla and Catharanthus roseus, homologous to a geissoschizine synthase (GS) cluster also uncovered.2025-01-01T00:00:00Z2025 Nov2026-06-05T16:45:47.105Z2026-05-18T03:13:12.46ZS-EPMC126475994129061110.1038/s41467-025-65543-z